Enhanced heterologous protein production in Pichia pastoris under increased air pressure
Autor(a) principal: | |
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Data de Publicação: | 2014 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/1822/31741 |
Resumo: | Pichia pastoris is a widely used host for the production of heterologous proteins. In this case, high cell densities are needed and oxygen is a major limiting factor. The increased air pressure could be used to improve the oxygen solubility in the medium and to reach the high oxygen demand of methanol metabolism. In this study, two P. pastoris strains producing two different recombinant proteins, one intracellular (β-galactosidase) and other extracellular (frutalin), were used to investigate the effect of increased air pressure on yeast growth in glycerol and heterologous protein production, using the methanol AOX1-inducible system. Experiments were carried out in a stainless steel bioreactor under total air pressure of 1 bar and 5 bar. The use of an air pressure raise of up to 5 bar proved to be applicable for P. pastoris cultivation. Moreover, no effects on the kinetic growth parameters and methanol utilization (Mut) phenotype of strains were found, while an increase in recombinant β-galactosidase-specific activity (ninefold) and recombinant frutalin production was observed. Furthermore, the air pressure raise led to a reduction in the secreted protease specific activity. This work shows for the first time that the application of an air pressure of 5 bar may be used as a strategy to decrease protease secretion and improve recombinant protein production in P. pastoris. |
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Enhanced heterologous protein production in Pichia pastoris under increased air pressureIncreased air pressureP. pastoris GS115P. pastoris KM71HRecombinant ß-galactosidaseRecombinant frutalinRecombinant β-galactosidaseScience & TechnologyPichia pastoris is a widely used host for the production of heterologous proteins. In this case, high cell densities are needed and oxygen is a major limiting factor. The increased air pressure could be used to improve the oxygen solubility in the medium and to reach the high oxygen demand of methanol metabolism. In this study, two P. pastoris strains producing two different recombinant proteins, one intracellular (β-galactosidase) and other extracellular (frutalin), were used to investigate the effect of increased air pressure on yeast growth in glycerol and heterologous protein production, using the methanol AOX1-inducible system. Experiments were carried out in a stainless steel bioreactor under total air pressure of 1 bar and 5 bar. The use of an air pressure raise of up to 5 bar proved to be applicable for P. pastoris cultivation. Moreover, no effects on the kinetic growth parameters and methanol utilization (Mut) phenotype of strains were found, while an increase in recombinant β-galactosidase-specific activity (ninefold) and recombinant frutalin production was observed. Furthermore, the air pressure raise led to a reduction in the secreted protease specific activity. This work shows for the first time that the application of an air pressure of 5 bar may be used as a strategy to decrease protease secretion and improve recombinant protein production in P. pastoris.The authors acknowledge the financial support provided by FCT (grant SFRH/BD/47371/2008 to Marlene Lopes and grant SFRH/BDP/63831/2009 to Carla Oliveira), by FCT Strategic Project PEst-OE/EQB/LA0023/2013 and by the Project "BioInd - Biotechnology and Bioengineering for improved Industrial and Agro-Food processes, REF. NORTE-07-0124-FEDER-000028" Co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER.American Chemical SocietyUniversidade do MinhoLopes, MarleneOliveira, Carla Cristina Marques deDomingues, LucíliaMota, M.Belo, Isabel20142014-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/31741eng8756-79381520-603310.1002/btpr.196425080319http://onlinelibrary.wiley.com/journal/10.1021/(ISSN)1520-6033info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-21T12:12:42Zoai:repositorium.sdum.uminho.pt:1822/31741Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:04:38.508995Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Enhanced heterologous protein production in Pichia pastoris under increased air pressure |
title |
Enhanced heterologous protein production in Pichia pastoris under increased air pressure |
spellingShingle |
Enhanced heterologous protein production in Pichia pastoris under increased air pressure Lopes, Marlene Increased air pressure P. pastoris GS115 P. pastoris KM71H Recombinant ß-galactosidase Recombinant frutalin Recombinant β-galactosidase Science & Technology |
title_short |
Enhanced heterologous protein production in Pichia pastoris under increased air pressure |
title_full |
Enhanced heterologous protein production in Pichia pastoris under increased air pressure |
title_fullStr |
Enhanced heterologous protein production in Pichia pastoris under increased air pressure |
title_full_unstemmed |
Enhanced heterologous protein production in Pichia pastoris under increased air pressure |
title_sort |
Enhanced heterologous protein production in Pichia pastoris under increased air pressure |
author |
Lopes, Marlene |
author_facet |
Lopes, Marlene Oliveira, Carla Cristina Marques de Domingues, Lucília Mota, M. Belo, Isabel |
author_role |
author |
author2 |
Oliveira, Carla Cristina Marques de Domingues, Lucília Mota, M. Belo, Isabel |
author2_role |
author author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Lopes, Marlene Oliveira, Carla Cristina Marques de Domingues, Lucília Mota, M. Belo, Isabel |
dc.subject.por.fl_str_mv |
Increased air pressure P. pastoris GS115 P. pastoris KM71H Recombinant ß-galactosidase Recombinant frutalin Recombinant β-galactosidase Science & Technology |
topic |
Increased air pressure P. pastoris GS115 P. pastoris KM71H Recombinant ß-galactosidase Recombinant frutalin Recombinant β-galactosidase Science & Technology |
description |
Pichia pastoris is a widely used host for the production of heterologous proteins. In this case, high cell densities are needed and oxygen is a major limiting factor. The increased air pressure could be used to improve the oxygen solubility in the medium and to reach the high oxygen demand of methanol metabolism. In this study, two P. pastoris strains producing two different recombinant proteins, one intracellular (β-galactosidase) and other extracellular (frutalin), were used to investigate the effect of increased air pressure on yeast growth in glycerol and heterologous protein production, using the methanol AOX1-inducible system. Experiments were carried out in a stainless steel bioreactor under total air pressure of 1 bar and 5 bar. The use of an air pressure raise of up to 5 bar proved to be applicable for P. pastoris cultivation. Moreover, no effects on the kinetic growth parameters and methanol utilization (Mut) phenotype of strains were found, while an increase in recombinant β-galactosidase-specific activity (ninefold) and recombinant frutalin production was observed. Furthermore, the air pressure raise led to a reduction in the secreted protease specific activity. This work shows for the first time that the application of an air pressure of 5 bar may be used as a strategy to decrease protease secretion and improve recombinant protein production in P. pastoris. |
publishDate |
2014 |
dc.date.none.fl_str_mv |
2014 2014-01-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/1822/31741 |
url |
http://hdl.handle.net/1822/31741 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
8756-7938 1520-6033 10.1002/btpr.1964 25080319 http://onlinelibrary.wiley.com/journal/10.1021/(ISSN)1520-6033 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
American Chemical Society |
publisher.none.fl_str_mv |
American Chemical Society |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
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1799132457053716480 |